Purification and characterization of an SDS-activated fibrinolytic enzyme from Eisenia fetida.
A sodium-dodecyl-sulfate-activated fibrinolytic enzyme from Eisenia fetida (the E. fetida enzyme) was purified by chromatography on DEAE Sepharose, Sephadex G-75, and Phenyl Sepharose 4. It (M(r) = 45 kDa) was composed of two subunits (M(r) = 26 kDa and M(r) = 18 kDa) held together by hydrophobic interactions. The enzyme displayed four activities when we used fibrin plates to detect the proteolytic activity. These were designated as CFPg (complete fibrinolysis in the plasminogen-rich plate), uCFPg (uncompleted fibrinolysis in the plasminogen-rich plate), CF (complete fibrinolysis in the plasminogen-free plate), and uCF (uncompleted fibrinolysis in the plasminogen-free plate). SDS activated CFPg and rendered the enzyme more sensitive to some inhibitors. Leupeptin, chymostatin, pepstatin, aprotinin, phenylmethylsulfonyl fluoride, and dithiothreitol had no effect on uCF. Pepstatin stimulated CFPg and uCFPg, while E-64, a thiol inhibitor, activated uCFPg and uCF. The N-terminal sequence of the large subunit was analyzed and compared with some known proteins. The large subunit alone had catalytic activity, while the small subunit did not. Using plasminogen as the substrate for defining peptide bond specificity, the E. fetida enzyme was observed to cleave the carboxyl side of basic amino acids, small neutral amino acids, and Met residue.
Yang
J S
JS
National Laboratory of Protein Engineering, College of Life Science, Peking University, Beijing, P.R. China. jsyang@perbms.pku.edu.cn
Ru
B G
BG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D010450:Endopeptidases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000737:chemistry*; D010958:Plasminogen / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D000596:Amino Acids; D005343:Fibrinolytic Agents; D011480:Protease Inhibitors; D012967:Sodium Dodecyl Sulfate; D010958:Plasminogen; D010450:Endopeptidases